MLK3, also known as SPRK, is a mitogen-activated protein kinase that can activate c-jun N- terminal kinase (JNK) and induce cell death in neuronal and non-neuronal cells. A broad-spectrum inhibitor of MLK family members (CEP-1347) is under clinical trials for treating neurodegenerative disorder related diseases like Parkinson's. TNF-a and ceramide have been shown to cause cell death during neurodegenerative disorders. Interestingly, ceramide has also emerged as a biochemical mediator of cell death in response to chemo- and radio- therapies. Studies from our laboratory have revealed that the cell death and differentiation inducing ligands, TNF-a and ceramide are potent agonists of MLK3 in mammalian cells. In addition, our more recent studies (both published and unpublished) indicate that (i) ceramides can directly associate, and activate MLK3, (ii) MLK3 mediates TNF-a and ceramide-induced JNK activation, (iii) TNF-a treatment of Jurkat cells triggers TRAF2 association with MLK3, (vi) overexpression of MLK3 causes cell death, and (v) Retinoic acid induced HL-60 cell differentiation is inhibited by MLKs specific inhibitor. Based on these observations, our major goals for this grant application are to elucidate the mechanisms of MLK3 activation by ceramide and TNF-a, and to define the physiological roles of ligand-activated MLK3 in cell death and differentiation pathways. These goals will be achieved following four specific aims. In aim 1, the in vitro and in vivo mechanisms of MLK3 activation by ceramide will be elucidated. In aim 2, the mechanism of TNF-a-induced MLK3 activation, and its synergy with ceramide-induced MLK3 activation will be examined. In aim 3, the physiological roles of MLK3 in mediating cell death and differentiation pathways will be elucidated using both biochemical and genetic approaches. In aim 4, the physiological role of MLK3 in neuronal apoptosis will be examined in MPTP mouse model (i.e. PD model), using recently created MLK3 KO and wild type animals. Elucidating the mechanism(s) of ceramide and TNF-ainduced MLK3 activation, and MLK3's role in modulating cell death and differentiation pathways will offer a basis for the development of targeted therapeutic interventions for pathologic neuronal loss occurring as a result of conditions, such as neurodegeneration, trauma or ischemia. Furthermore, the results from these studies will aide in the development of novel pharmacological approaches towards increasing the efficiency and specificity of chemo- or radio- sensitization for cancer therapies.